Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification
Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification
复制标题
DOI：
10.1074/jbc.m410242200
复制
发表时间：
2005-01-28
影响因子：
4.8
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Humphries, KM;Deal, MS;Taylor, SS
cAMP依赖性蛋白激酶（PKA）的催化亚基在苏氨酸197和丝氨酸338处磷酸化。位于活化环中的苏氨酸197的磷酸化是协调活性位点构象和最佳酶活性所必需的。然而，由于磷酸化的表观组成性质和激酶对磷酸酶处理的一般抗性，这种磷酸化尚未被广泛理解为调节位点。我们在这里证明，所观察到的催化亚基去磷酸化的阻力，部分是由于高度亲核的半胱氨酸199位于近端的磷酸对苏氨酸197的存在。在体外进行的实验表明，突变（半胱氨酸199丙氨酸），氧化，如谷胱甘肽化或内部二硫键的形成，或烷基化的C-亚基增强其被脱磷酸化的能力。此外，PDK 1对还原的C亚基的再磷酸化产生了一个循环，从而可以重新激活失活的激酶。为了证明PKA的巯基修饰可以导致体内增强的去磷酸化，用N-乙基马来酰亚胺（NEM）处理PC 12细胞。这种处理导致PKA完全失活和苏氨酸197的去磷酸化。NEM的这种作用取决于用PKA激活剂预先处理细胞，证明全酶对硫醇烷基化介导的去磷酸化的抗性。我们的研究结果还表明，NEM处理的PC 12细胞增强了蛋白激酶Calpha激活环的去磷酸化，这表明AGC家族激酶的成员之间的共同调节机制。
The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.